Porphyromonas gingivalis is a periodontal pathogen implicated in the initiation and progression of chronic periodontitis in adults. This organism produces major classes of biologically active sphingolipids, termed phosphorylated dihydroceramides and other unusual lipids. These lipids are potent inflammatory and immune cell activators. The primary goal of this proposal is to evaluate how these lipids promote bone loss associated with periodontitis. Another major goal is to understand how these lipids mediate their effects on bone through engagement of the innate immune system, specifically through Toll Receptor 2 (TLR2) engagement. Understanding how these lipids promote TLR2-dependent bone loss is relevant specifically to the reported effects of P. gingivalis on periodontal bone loss in experimental animals, and may be important in promotion of bone loss in rheumatoid arthritis associated with periodontal disease. P. gingivalis lipids contaminate diseased periodontal tissues in such a way that direct bacterial invasion does not account for the observed bacterial lipids recovered. This application proposes to quantify bacterial lipid levels in diseased tissue samples, using collisional mass spectrometry, in order to determine the appropriate types and mixtures of bacterial lipids for testing in bone cell cultures and experimental animals. Those lipids recovered in diseased periodontal tissues will be tested in cell culture for their capacity to either inhibit bone formation or activate bone resorption as reflected by specific changes in gene expression, increased secretion of bone destructive cytokines and direct activation of cells that mediate bone resorption. Furthermore, bone loss will be evaluated in vivo by administering bacterial lipids to bone surfaces, followed by evaluation of bone loss and the associated alterations in either bone forming or bone resorbing cells. Finally we will examine the role of TLR2 in these processes by comparing bacterial lipid effects in bone cells isolated from either wild type or TLR2 knockout animals or by testing lipids directly in these animals. The experiments summarized in this proposal are critical to explaining how P. gingivalis promotes bone loss in periodontal diseases and may provide another mechanistic explanation for bone loss associated with common systemic diseases such as rheumatoid arthritis.